【作者】 柯玉文；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2007， 博士

【摘要】 细胞分裂周期是由时空上高度精密调节的蛋白相互作用信号途径所协调的，从而保证子代细胞从母代得到准确的分成两个相同的等分的遗传信息。细胞周期机制在时间与空间上高度协调控制，一旦这些精确调控的丢失则会丧失基因组稳定性和细胞的可塑性，从而增加癌症发生的风险。激酶信号途径调节有丝分裂纺锤体的动态性与可塑性以确保准确的染色体分离。有丝分裂启动的关键因子是cyclin-dependent kinase，Cdc2。此外，还有三类serine／threonine激酶家族在有丝分裂中起了重要的调节作用：Polo kinases，Aurora kinases，the NIMA-related kinases。在多种癌症中过表达的Aurora激酶家族参与调节中心体成熟，纺锤体组装，染色体分离和胞质分裂。我博士期间的研究工作主要集中在Aurora激酶信号途径对有丝分裂纺锤体动态性与可塑性的功能调节。在这里，我们首次对在纺锤体可塑性起关键作用的新中心体结合蛋白ATIP3进行鉴定与功能性描述。使用单一识别的抗体，我的免疫荧光结果表明内源性ATIP3在进入分裂期定位在中心体上，胞质分裂时转移到中体上。外源性ATIP3与微管共定位，缺失突变分析表明489-762残基负责ATIP3与微管结合。通过RNA干扰抑制ATIP3的表达会导致严重的有丝分裂差错：多极纺锤体、不正常的中心粒结构、细胞周期阻断、甚至凋亡。生化试验结果显示ATIP3能与Aurora-A在体内体外相互作用，暗示ATIP3的磷酸化调节在双极纺锤体组装和有丝分裂进程起了重要的作用。Survivin作为一个内层着丝粒蛋白不仅参与调节染色体分离和胞质分裂，还参与抑制细胞凋亡。survivin在很多肿瘤中过表达，揭示了它的抗调亡功能在肿瘤发生中的可能性作用。为了阐明现阶段对有丝分裂纺锤体结合的survivin如何决定细胞命运的模糊认识，我进行了系统的突变分析，结果令人惊奇：survivin在有丝分裂结构上的分布独立于它在细胞命运决定中的规律。另外，这些突变体都具有与Aurora-B结合的能力并且不影响Aurora-B和CENP-E在动点和中体的定位。而过表达survivin促凋亡点突变体的细胞可以顺利完成细胞分裂，但在进入下一个间期时走向凋亡。我们相信survivin在有丝分裂中的调节功能在时间和空间上都与它的抑制细胞凋亡的功能相互独立。我在对Aurora-B分子功能进行研究时鉴定了一个新的Aurora-B结合蛋白：p53。肿瘤抑制子p53在应对多种细胞胁迫过程中决定细胞生长或死亡中起了重要的作用。我的生化试验表明发现Aurora-B可以磷酸化p53 Ser315位残基。有趣的是，在293T细胞中表达时，非磷酸化突变体p53^S315A比模拟磷酸化突变体p53^S315D具有更高的稳定性。因此，我的研究首次证明肿瘤抑制子p53在时间与空间上被CDK2，Aurora-A，Aurora-B所调节。综上所述，我们的研究阐明了Aurora激酶与它们的底物ATIP3、survivin、p53的相互作用规律，为进一步认识Aurora激酶信号通路对细胞有丝分裂纺锤体动态性与可塑性的调节机理提供了新思路。更多还原

【Abstract】 The cell division cycle is orchestrated by a cascade of spatiotemporal regulated protein interaction, which ensures a faithful segregation of parent genome into two identical copies for equal distribution into two daughter cells. Any perturbation of this highly coordinated temporal and spatial control of cell cycle machinery will lead to a compromised genomic stability and cellular plasticity, which contributes to the tumorigenesis.Mitosis is driven by kinase cascades that govern mitotic spindle dynamics and plasticity to ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase, Cdc2. In addition to the master mitotic kinase Cdc2, there are three protein serine/threonine kinase families, the Polo kinases, Aurora kinases and the NIMA-related kinases. Aurora kinases regulate centrosome maturation, spindle assembly, chromosome segregation and cytokinesis, which overexpress in a variety of tumors. My doctoral project is centered on the functional delineation of Aurora kinase cascade in mitotic spindle dynamics and plasticity.Here, we present my identification and functional characterization of a novel centrosome-associated protein ATIP3 essential for mitotic spindle plasticity. Using mono-specific antibody, my immunofluorescence microscopic analysis demonstrated that endogenous ATIP3 is localized at centrosome at the onset of mitosis, and relocated to the midbody during cytokinesis, while exogenous ATIP3 localized to MTs. My deletion analysis revealed that the region containing 489-762aa is essential for ATIP3 binding to microtubule. Inhibition of ATIP3 by RNA interference resulted in severe mitotic defects: multi-polar spindles, abnormal centrioles, mitotic delays, and apoptosis. Our biochemical characterization revealed that ATIP3 is a substrate of Aurora-A in vitro and in vivo, indicating the phospho-regulation of ATIP3 plays an important role in the assembly of a bipolar spindle and in orchestration of mitotic progression.Survivin is an inner centromere protein implicated in cell division and apoptotic decision. Survivin is overexpressed in most solid tumor, raising the possibility of its anti-apoptotic function in tumorigenesis. To clarify the confusion in the literature as to the functional relevance of survivin in cell fate decision when it is associated with the mitotic spindle, I conducted a systemic mutational analysis. To my surprise, its distribution to the mitotic apparatus is independent of its role in cell fate decision. In addition, those survivin mutants bear Aurora-B-binding activity and did not perturb the localization of Aurora-B and CENP-E to either kinetochore of midbody. In addition, cells overexpressing these survivin mutants progressed throughout mitosis completely, and committed apoptosis in the next interphase. We believe that the function of survivin in mitosis is spatio-temporally separated from its role in apoptosis.My pursuit for molecular function of Aurora-B led to the identification of a novel Aurora-B-binding partner p53. The tumor suppressor p53 plays an important role in the decision of cell growth or death in response to a variety of stimuli. My biochemical characterizeation shows that p53 is phosphorylated by Aurora-B at Ser315. Interestingly, the non-phosphorylatable mutant of p53^S315A bears an increased stability compared to that of phosphor-mimicking mutant p53^S315D when they were expressed in 293T cells. Thus, my studies provide the first line evidence in which tumor suppressor p53 is regulated by CDK2, Aurora-A and Aurora-B, in a spatial and temporal manner.Taken together, my studies illustrate functional interplay between Aurora kinases and their cognate substrates such as ATIP3, p53 and survivin, which provide novel insights into a better understanding of Aurora kinase cascade in mitotic spindle dynamics and plasticity.更多还原